1. Field of the Invention
The present invention relates generally to the field of immunology and, more particularly, the present invention relates to an expression vector for the display of lamprey VLR proteins on a yeast cell surface for selection of high affinity proteins for use in combinatorial libraries.
2. Description of the Related Art Technical Field
At least 10,000 antibodies (Abs) are commercially available for biomedical and biotechnology applications. Abs are used in various medical applications including in vivo diagnostics; as inhibitors of extracellular proteases; as antagonists of cellular receptors or soluble factors; as tissue-targeting vehicles that deliver linked toxic molecules, cytokines or enzymes; or as scavengers of toxic compounds (28). In 2007, the FDA approved 21 Ab-based biopharmaceuticals, mainly for treatment of cancer and autoimmune diseases, approached annual sales of $20 billion (46). Other biotechnology applications for Abs include detection reagents in immunochemical assays such as ELISA, immunoprecipitation, Western blotting, flow cytometry, as biosensors in chip technology and for separation of analytes.
The application of recombinant Abs, as recognition molecules has pitfalls because immunoglobulins (Igs) are large molecules consisting of light and heavy chains linked by disulfide bonds, which requires elaborate and expensive steps for recombinant expression. The complex architecture of the antigen-binding site, formed by six hypervariable loops, three from each chain, further complicates the manipulations required for generation of synthetic Ab libraries. In addition, subunits of Ab fragments often dissociate, or tend to aggregate when fused to effector domains. These technical limitations, as well as major patent restrictions, have driven an intensive search for alternatives to Abs in the form of synthetic binding scaffolds such as ankyrin repeats, fibronectins, lipocalins, protein A, src homology domains and thioredoxin (27, 28). But none of these are natural antigen receptors.
Jawed vertebrates, such as sharks, birds, and mammals, mount a robust humoral response on immune stimulation with foreign antigens. Typically, naïve B lymphocytes bind antigens with low affinity via surface IgM. Subsequently, antibody genes undergo somatic hypermutation, and those clones with highest affinity are selected to produce effective immune responses and form the memory pool (1, 2). Lamprey and hagfish are jawless fish, representatives of the ancestral vertebrate taxon, which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of the Ig superfamily domains found in Ig-based antibodies and T cell receptors (TCRs), the variable lymphocyte receptors (VLRs) of lamprey and hagfish consist of highly diverse leucine-rich repeat (LRR) modules (3, 6). LRRs are ancient protein modules that are prevalent building blocks of animal and plant pattern recognition molecules, such as Toll and Toll-like receptors, nucleotide oligomerization domain (NOD), LRRs, and plant disease-resistance genes, which are triggered by an exceptionally diverse array of ligands (7). Interestingly, however, VLRs are not related to these pattern recognition molecules, but instead are closely related to the vertebrate-specific von Willebrand factor receptor GpIbα, a member of the family of platelet LRR-containing hemostatic receptors (6). Jawless vertebrates thus evolved their rearranging antigen receptors from LRR scaffolds, elaborating the only known adaptive immune system not based on Ig or on TCR (6).
However, little is known about the antigen-binding properties of VLRs, or about how the naïve VLR repertoire develops into a protective shield in immune-stimulated animals. Thus it would be advantageous to use the natural antigen antigen-binding properties of VLRs to develop systems and methods for diagnostic applications, immunoaffinity purification, and engineering novel fusion proteins that do not include the shortcomings of Ig antibodies.